Enhanced polyubiquitination of Shank3 and NMDA receptor in a mouse model of autism.

We have created a mouse genetic model that mimics a human mutation of Shank3 that deletes the C terminus and is associated with autism. Expressed as a single copy [Shank3(+/ΔC) mice], Shank3ΔC protein interacts with the wild-type (WT) gene product and results in >90% reduction of Shank3 at synapses. This "gain-of-function" phenotype is linked to increased polyubiquitination of WT Shank3 and its redistribution into proteasomes. Similarly, the NR1 subunit of the NMDA receptor is reduced at synapses with increased polyubiquitination. Assays of postsynaptic density proteins, spine morphology, and synapse number are unchanged in Shank3(+/ΔC) mice, but the amplitude of NMDAR responses is reduced together with reduced NMDAR-dependent LTP and LTD. Reciprocally, mGluR-dependent LTD is markedly enhanced. Shank3(+/ΔC) mice show behavioral deficits suggestive of autism and reduced NMDA receptor function. These studies reveal a mechanism distinct from haploinsufficiency by which mutations of Shank3 can evoke an autism-like disorder.

Arc/Arg3.1 regulates an endosomal pathway essential for activity-dependent ß-amyloid generation.

Assemblies of ß-amyloid (Aß) peptides are pathological mediators of Alzheimer's Disease (AD) and are produced by the sequential cleavages of amyloid precursor protein (APP) by ß-secretase (BACE1) and γ-secretase. The generation of Aß is coupled to neuronal activity, but the molecular basis is unknown. Here, we report that the immediate early gene Arc is required for activity-dependent generation of Aß. Arc is a postsynaptic protein that recruits endophilin2/3 and dynamin to early/recycling endosomes that traffic AMPA receptors to reduce synaptic strength in both hebbian and non-hebbian forms of plasticity. The Arc-endosome also traffics APP and BACE1, and Arc physically associates with presenilin1 (PS1) to regulate γ-secretase trafficking and confer activity dependence. Genetic deletion of Arc reduces Aß load in a transgenic mouse model of AD. In concert with the finding that patients with AD can express anomalously high levels of Arc, we hypothesize that Arc participates in the pathogenesis of AD.

The BAD-BAX-Caspase-3 Cascade Modulates Synaptic Vesicle Pools via Autophagy.

The BAD-BAX-caspase-3 cascade is a canonical apoptosis pathway. Macroautophagy ("autophagy" hereinafter) is a process by which organelles and aggregated proteins are delivered to lysosomes for degradation. Here, we report a new function of the BAD-BAX-caspase-3 cascade and autophagy in the control of synaptic vesicle pools. We found that, in hippocampal neurons of male mice, the BAD-BAX-caspase-3 pathway regulates autophagy, which in turn limits the size of synaptic vesicle pools and influences the kinetics of activity-induced depletion and recovery of synaptic vesicle pools. Moreover, the caspase-autophagy pathway is engaged by fear conditioning to facilitate associative fear learning and memory. This work identifies a new mechanism for controlling synaptic vesicle pools, and a novel, nonapoptotic, presynaptic function of the BAD-BAX-caspase-3 cascade.SIGNIFICANCE STATEMENT Despite the importance of synaptic vesicles for neurons, little is known about how the size of synaptic vesicle pools is maintained under basal conditions and regulated by neural activity. This study identifies a new mechanism for the control of synaptic vesicle pools, and a new, nonapoptotic function of the BAD-BAX-caspase-3 pathway in presynaptic terminals. Additionally, it indicates that autophagy is not only a homeostatic mechanism to maintain the integrity of cells and tissues, but also a process engaged by neural activity to regulate synaptic vesicle pools for optimal synaptic responses, learning, and memory.

Alzheimer's disease/dementia-associated brain pathology in aging DPP6-KO mice.

We have previously reported that the single transmembrane protein Dipeptidyl Peptidase Like 6 (DPP6) impacts neuronal and synaptic development. DPP6-KO mice are impaired in hippocampal-dependent learning and memory and exhibit smaller brain size. Recently, we have described novel structures in hippocampal area CA1 in aging mice, apparently derived from degenerating presynaptic terminals, that are significantly more prevalent in DPP6-KO mice compared to WT mice of the same age and that these structures were observed earlier in development in DPP6-KO mice. These novel structures appear as clusters of large puncta that colocalize NeuN, synaptophysin, and chromogranin A, and also partially label for MAP2, amyloid ß, APP, α-synuclein, and phosphorylated tau, with synapsin-1 and VGluT1 labeling on their periphery. In this current study, using immunofluorescence and electron microscopy, we confirm that both APP and amyloid ß are prevalent in these structures; and we show with immunofluorescence the presence of similar structures in humans with Alzheimer's disease. Here we also found evidence that aging DPP6-KO mutants show additional changes related to Alzheimer's disease. We used in vivo MRI to show reduced size of the DPP6-KO brain and hippocampus. Aging DPP6-KO hippocampi contained fewer total neurons and greater neuron death and had diagnostic biomarkers of Alzheimer's disease present including accumulation of amyloid ß and APP and increase in expression of hyper-phosphorylated tau. The amyloid ß and phosphorylated tau pathologies were associated with neuroinflammation characterized by increases in microglia and astrocytes. And levels of proinflammatory or anti-inflammatory cytokines increased in aging DPP6-KO mice. We finally show that aging DPP6-KO mice display circadian dysfunction, a common symptom of Alzheimer's disease. Together these results indicate that aging DPP6-KO mice show symptoms of enhanced neurodegeneration reminiscent of dementia associated with a novel structure resulting from synapse loss and neuronal death. This study continues our laboratory's work in discerning the function of DPP6 and here provides compelling evidence of a direct role of DPP6 in Alzheimer's disease.

Myosin 18Aα targets the guanine nucleotide exchange factor ß-Pix to the dendritic spines of cerebellar Purkinje neurons and promotes spine maturation.

Myosin 18Aα is a myosin 2-like protein containing unique N- and C-terminal protein interaction domains that co-assembles with myosin 2. One protein known to bind to myosin 18Aα is ß-Pix, a guanine nucleotide exchange factor (GEF) for Rac1 and Cdc42 that has been shown to promote dendritic spine maturation by activating the assembly of actin and myosin filaments in spines. Here, we show that myosin 18A⍺ concentrates in the spines of cerebellar Purkinje neurons via co-assembly with myosin 2 and through an actin binding site in its N-terminal extension. miRNA-mediated knockdown of myosin 18A⍺ results in a significant defect in spine maturation that is rescued by an RNAi-immune version of myosin 18A⍺. Importantly, ß-Pix co-localizes with myosin 18A⍺ in spines, and its spine localization is lost upon myosin 18A⍺ knockdown or when its myosin 18A⍺ binding site is deleted. Finally, we show that the spines of myosin 18A⍺ knockdown Purkinje neurons contain significantly less F-actin and myosin 2. Together, these data argue that mixed filaments of myosin 2 and myosin 18A⍺ form a complex with ß-Pix in Purkinje neuron spines that promotes spine maturation by enhancing the assembly of actin and myosin filaments downstream of ß-Pix's GEF activity.

Dysbindin-1 regulates mitochondrial fission and gamma oscillations.

Mitochondria are cellular ATP generators. They are dynamic structures undergoing fission and fusion. While much is known about the mitochondrial fission machinery, the mechanism of initiating fission and the significance of fission to neurophysiology are largely unclear. Gamma oscillations are synchronized neural activities that impose a great energy challenge to synapses. The cellular mechanism of fueling gamma oscillations has yet to be defined. Here, we show that dysbindin-1, a protein decreased in the brain of individuals with schizophrenia, is required for neural activity-induced fission by promoting Drp1 oligomerization. This process is engaged by gamma-frequency activities and in turn, supports gamma oscillations. Gamma oscillations and novel object recognition are impaired in dysbindin-1 null mice. These defects can be ameliorated by increasing mitochondrial fission. These findings identify a molecular mechanism for activity-induced mitochondrial fission, a role of mitochondrial fission in gamma oscillations, and mitochondrial fission as a potential target for improving cognitive functions.

The phenotypic landscape of a Tbc1d24 mutant mouse includes convulsive seizures resembling human early infantile epileptic encephalopathy.

Epilepsy, deafness, onychodystrophy, osteodystrophy and intellectual disability are associated with a spectrum of mutations of human TBC1D24. The mechanisms underlying TBC1D24-associated disorders and the functions of TBC1D24 are not well understood. Using CRISPR-Cas9 genome editing, we engineered a mouse with a premature translation stop codon equivalent to human S324Tfs*3, a recessive mutation of TBC1D24 associated with early infantile epileptic encephalopathy (EIEE). Homozygous S324Tfs*3 mice have normal auditory and vestibular functions but show an abrupt onset of spontaneous seizures at postnatal day 15 recapitulating human EIEE. The S324Tfs*3 variant is located in an alternatively spliced micro-exon encoding six perfectly conserved amino acids incorporated postnatally into TBC1D24 protein due to a micro-exon utilization switch. During embryonic and early postnatal development, S324Tfs*3 homozygotes produce predominantly the shorter wild-type TBC1D24 protein isoform that omits the micro-exon. S324Tfs*3 homozygotes show an abrupt onset of seizures at P15 that correlates with a developmental switch to utilization of the micro-exon. A mouse deficient for alternative splice factor SRRM3 impairs incorporation of the Tbc1d24 micro-exon. Wild-type Tbc1d24 mRNA is abundantly expressed in the hippocampus using RNAscope in situ hybridization. Immunogold electron microscopy using a TBC1D24-specific antibody revealed that TBC1D24 is associated with clathrin-coated vesicles and synapses of hippocampal neurons, suggesting a crucial role of TBC1D24 in vesicle trafficking important for neuronal signal transmission. This is the first characterization of a mouse model of human TBC1D24-associated EIEE that can now be used to screen for antiepileptogenic drugs ameliorating TBCID24 seizure disorders.

Neurolastin, a dynamin family GTPase, translocates to mitochondria upon neuronal stress and alters mitochondrial morphology in vivo.

Neurolastin is a dynamin family GTPase that also contains a RING domain and exhibits both GTPase and E3 ligase activities. It is specifically expressed in the brain and is important for synaptic transmission, as neurolastin knockout animals have fewer dendritic spines and exhibit a reduction in functional synapses. Our initial study of neurolastin revealed that it is membrane-associated and partially co-localizes with endosomes. Using various biochemical and cell-culture approaches, we now show that neurolastin also localizes to mitochondria in HeLa cells, cultured neurons, and brain tissue. We found that the mitochondrial localization of neurolastin depends upon an N-terminal mitochondrial targeting sequence and that neurolastin is imported into the mitochondrial intermembrane space. Although neurolastin was only partially mitochondrially localized at steady state, it displayed increased translocation to mitochondria in response to neuronal stress and mitochondrial fragmentation. Interestingly, inactivation or deletion of neurolastin's RING domain also increased its mitochondrial localization. Using EM, we observed that neurolastin knockout animals have smaller but more numerous mitochondria in cerebellar Purkinje neurons, indicating that neurolastin regulates mitochondrial morphology. We conclude that the brain-specific dynamin GTPase neurolastin exhibits stress-responsive localization to mitochondria and is required for proper mitochondrial morphology.

Enlargement of Ribbons in Zebrafish Hair Cells Increases Calcium Currents But Disrupts Afferent Spontaneous Activity and Timing of Stimulus Onset.

In sensory hair cells of auditory and vestibular organs, the ribbon synapse is required for the precise encoding of a wide range of complex stimuli. Hair cells have a unique presynaptic structure, the synaptic ribbon, which organizes both synaptic vesicles and calcium channels at the active zone. Previous work has shown that hair-cell ribbon size is correlated with differences in postsynaptic activity. However, additional variability in postsynapse size presents a challenge to determining the specific role of ribbon size in sensory encoding. To selectively assess the impact of ribbon size on synapse function, we examined hair cells in transgenic zebrafish that have enlarged ribbons, without postsynaptic alterations. Morphologically, we found that enlarged ribbons had more associated vesicles and reduced presynaptic calcium-channel clustering. Functionally, hair cells with enlarged ribbons had larger global and ribbon-localized calcium currents. Afferent neuron recordings revealed that hair cells with enlarged ribbons resulted in reduced spontaneous spike rates. Additionally, despite larger presynaptic calcium signals, we observed fewer evoked spikes with longer latencies from stimulus onset. Together, our work indicates that hair-cell ribbon size influences the spontaneous spiking and the precise encoding of stimulus onset in afferent neurons.SIGNIFICANCE STATEMENT Numerous studies support that hair-cell ribbon size corresponds with functional sensitivity differences in afferent neurons and, in the case of inner hair cells of the cochlea, vulnerability to damage from noise trauma. Yet it is unclear whether ribbon size directly influences sensory encoding. Our study reveals that ribbon enlargement results in increased ribbon-localized calcium signals, yet reduces afferent spontaneous activity and disrupts the timing of stimulus onset, a distinct aspect of auditory and vestibular encoding. These observations suggest that varying ribbon size alone can influence sensory encoding, and give further insight into how hair cells transduce signals that cover a wide dynamic range of stimuli.

Ciliary proteins Bbs8 and Ift20 promote planar cell polarity in the cochlea.

Primary cilia have been implicated in the generation of planar cell polarity (PCP). However, variations in the severity of polarity defects in different cilia mutants, coupled with recent demonstrations of non-cilia-related actions of some cilia genes, make it difficult to determine the basis of these polarity defects. To address this issue, we evaluated PCP defects in cochlea from a selection of mice with mutations in cilia-related genes. Results indicated notable PCP defects, including mis-oriented hair cell stereociliary bundles, in Bbs8 and Ift20 single mutants that are more severe than in other cilia gene knockouts. In addition, deletion of either Bbs8 or Ift20 results in disruptions in asymmetric accumulation of the core PCP molecule Vangl2 in cochlear cells, suggesting a role for Bbs8 and/or Ift20, possibly upstream of core PCP asymmetry. Consistent with this, co-immunoprecipitation experiments indicate direct interactions of Bbs8 and Ift20 with Vangl2. We observed localization of Bbs and Ift proteins to filamentous actin as well as microtubules. This could implicate these molecules in selective trafficking of membrane proteins upstream of cytoskeletal reorganization, and identifies new roles for cilia-related proteins in cochlear PCP.

Dendrosomatic Sonic Hedgehog Signaling in Hippocampal Neurons Regulates Axon Elongation.

The presence of Sonic Hedgehog (Shh) and its signaling components in the neurons of the hippocampus raises a question about what role the Shh signaling pathway may play in these neurons. We show here that activation of the Shh signaling pathway stimulates axon elongation in rat hippocampal neurons. This Shh-induced effect depends on the pathway transducer Smoothened (Smo) and the transcription factor Gli1. The axon itself does not respond directly to Shh; instead, the Shh signal transduction originates from the somatodendritic region of the neurons and occurs in neurons with and without detectable primary cilia. Upon Shh stimulation, Smo localization to dendrites increases significantly. Shh pathway activation results in increased levels of profilin1 (Pfn1), an actin-binding protein. Mutations in Pfn1's actin-binding sites or reduction of Pfn1 eliminate the Shh-induced axon elongation. These findings indicate that Shh can regulate axon growth, which may be critical for development of hippocampal neurons. SIGNIFICANCE STATEMENT: Although numerous signaling mechanisms have been identified that act directly on axons to regulate their outgrowth, it is not known whether signals transduced in dendrites may also affect axon outgrowth. We describe here a transcellular signaling pathway in embryonic hippocampal neurons in which activation of Sonic Hedgehog (Shh) receptors in dendrites stimulates axon growth. The pathway involves the dendritic-membrane-associated Shh signal transducer Smoothened (Smo) and the transcription factor Gli, which induces the expression of the gene encoding the actin-binding protein profilin 1. Our findings suggest scenarios in which stimulation of Shh in dendrites results in accelerated outgrowth of the axon, which therefore reaches its presumptive postsynaptic target cell more quickly. By this mechanism, Shh may play critical roles in the development of hippocampal neuronal circuits.

Two N-glycosylation Sites in the GluN1 Subunit Are Essential for Releasing N-methyl-d-aspartate (NMDA) Receptors from the Endoplasmic Reticulum.

NMDA receptors (NMDARs) comprise a subclass of neurotransmitter receptors whose surface expression is regulated at multiple levels, including processing in the endoplasmic reticulum (ER), intracellular trafficking via the Golgi apparatus, internalization, recycling, and degradation. With respect to early processing, NMDARs are regulated by the availability of GluN subunits within the ER, the presence of ER retention and export signals, and posttranslational modifications, including phosphorylation and palmitoylation. However, the role of N-glycosylation, one of the most common posttranslational modifications, in regulating NMDAR processing has not been studied in detail. Using biochemistry, confocal and electron microscopy, and electrophysiology in conjunction with a lentivirus-based molecular replacement strategy, we found that NMDARs are released from the ER only when two asparagine residues in the GluN1 subunit (Asn-203 and Asn-368) are N-glycosylated. Although the GluN2A and GluN2B subunits are also N-glycosylated, their N-glycosylation sites do not appear to be essential for surface delivery of NMDARs. Furthermore, we found that removing N-glycans from native NMDARs altered the receptor affinity for glutamate. Our results suggest a novel mechanism by which neurons ensure that postsynaptic membranes contain sufficient numbers of functional NMDARs.

Cholinergic modulation of large-conductance calcium-activated potassium channels regulates synaptic strength and spine calcium in cartwheel cells of the dorsal cochlear nucleus.

Acetylcholine is a neuromodulatory transmitter that controls synaptic plasticity and sensory processing in many brain regions. The dorsal cochlear nucleus (DCN) is an auditory brainstem nucleus that integrates auditory signals from the cochlea with multisensory inputs from several brainstem nuclei and receives prominent cholinergic projections. In the auditory periphery, cholinergic modulation serves a neuroprotective function, reducing cochlear output under high sound levels. However, the role of cholinergic signaling in the DCN is less understood. Here we examine postsynaptic mechanisms of cholinergic modulation at glutamatergic synapses formed by parallel fiber axons onto cartwheel cells (CWCs) in the apical DCN circuit from mouse brainstem slice using calcium (Ca) imaging combined with two-photon laser glutamate uncaging onto CWC spines. Activation of muscarinic acetylcholine receptors (mAChRs) significantly increased the amplitude of both uncaging-evoked EPSPs (uEPSPs) and spine Ca transients. Our results demonstrate that mAChRs in CWC spines act by suppressing large-conductance calcium-activated potassium (BK) channels, and this effect is mediated through the cAMP/protein kinase A signaling pathway. Blocking BK channels relieves voltage-dependent magnesium block of NMDA receptors, thereby enhancing uEPSPs and spine Ca transients. Finally, we demonstrate that mAChR activation inhibits L-type Ca channels and thus may contribute to the suppression of BK channels by mAChRs. In summary, we demonstrate a novel role for BK channels in regulating glutamatergic transmission and show that this mechanism is under modulatory control of mAChRs.

Neto auxiliary protein interactions regulate kainate and NMDA receptor subunit localization at mossy fiber-CA3 pyramidal cell synapses.

Neto1 and Neto2 auxiliary subunits coassemble with NMDA receptors (NMDARs) and kainate receptors (KARs) to modulate their function. In the hippocampus, Neto1 enhances the amplitude and prolongs the kinetics of KAR-mediated currents at mossy fiber (MF)-CA3 pyramidal cell synapses. However, whether Neto1 trafficks KARs to synapses or simply alters channel properties is unresolved. Therefore, postembedding electron microscopy was performed to investigate the localization of GluK2/3 subunits at MF-CA3 synapses in Neto-null mice. Postsynaptic GluK2/3 Immunogold labeling was substantially reduced in Neto-null mice compared with wild types. Moreover, spontaneous KAR-mediated synaptic currents and metabotropic KAR signaling were absent in CA3 pyramidal cells of Neto-null mice. A similar loss of ionotropic and metabotropic KAR function was observed in Neto1, but not Neto2, single knock-out mice, specifically implicating Neto1 in regulating CA3 pyramidal cell KAR localization and function. Additional controversy pertains to the role of Neto proteins in modulating synaptic NMDARs. While Immunogold labeling for GluN2A at MF-CA3 synapses was comparable between wild-type and Neto-null mice, labeling for postsynaptic GluN2B was robustly increased in Neto-null mice. Accordingly, NMDAR-mediated currents at MF-CA3 synapses exhibited increased sensitivity to a GluN2B-selective antagonist in Neto1 knockouts relative to wild types. Thus, despite preservation of the overall MF-CA3 synaptic NMDAR-mediated current, loss of Neto1 alters NMDAR subunit composition. These results confirm that Neto protein interactions regulate synaptic localization of KAR and NMDAR subunits at MF-CA3 synapses, with implications for both ionotropic and metabotropic glutamatergic recruitment of the CA3 network.

Cytoskeletal changes in actin and microtubules underlie the developing surface mechanical properties of sensory and supporting cells in the mouse cochlea.

Correct patterning of the inner ear sensory epithelium is essential for the conversion of sound waves into auditory stimuli. Although much is known about the impact of the developing cytoskeleton on cellular growth and cell shape, considerably less is known about the role of cytoskeletal structures on cell surface mechanical properties. In this study, atomic force microscopy (AFM) was combined with fluorescence imaging to show that developing inner ear hair cells and supporting cells have different cell surface mechanical properties with different developmental time courses. We also explored the cytoskeletal organization of developing sensory and non-sensory cells, and used pharmacological modulation of cytoskeletal elements to show that the developmental increase of hair cell stiffness is a direct result of actin filaments, whereas the development of supporting cell surface mechanical properties depends on the extent of microtubule acetylation. Finally, this study found that the fibroblast growth factor signaling pathway is necessary for the developmental time course of cell surface mechanical properties, in part owing to the effects on microtubule structure.

An essential role for inhibitor-2 regulation of protein phosphatase-1 in synaptic scaling.

Protein phosphatase-1 (PP1) activity is important for many calcium-dependent neuronal functions including Hebbian synaptic plasticity and learning and memory. PP1 activity is necessary for the induction of long-term depression, whereas downregulation of PP1 activity is required for the normal induction of long-term potentiation. However, how PP1 is activated is not clear. Moreover, it is not known whether PP1 plays a role in homeostatic synaptic scaling, another form of synaptic plasticity which functions to reset the neuronal firing rate in response to chronic neuronal activity perturbations. In this study, we found that PP1 inhibitor-2 (I-2) is phosphorylated at serine 43 (S43) in rat and mouse cortical neurons in response to bicuculine application. Expression of I-2 phosphorylation-blocking mutant I-2 (S43A) blocked the dephosphorylation of GluA2 at serine 880, AMPA receptor trafficking, and synaptic downscaling induced by bicuculline application. Our data suggest that the phosphorylation of I-2 at S43 appears to be mediated by L-type calcium channels and calcium/calmodulin-dependent myosin light-chain kinase. Our work thus reveals a novel calcium-induced PP1 activation pathway critical for homeostatic synaptic plasticity.

Sonic hedgehog regulates presynaptic terminal size, ultrastructure and function in hippocampal neurons.

Sonic hedgehog (Shh) signaling is essential to the patterning of the embryonic neural tube, but its presence and function in the postmitotic differentiated neurons in the brain remain largely uncharacterized. We recently showed that Shh and its signaling components, Patched and Smoothened, are expressed in postnatal and adult hippocampal neurons. We have now examined whether Shh signaling has a function in these neurons. Using cultured hippocampal neurons as a model system, we found that presynaptic terminals become significantly larger in response to the application of Shh. Ultrastructural examination confirmed the enlarged presynaptic profiles and also revealed variable increases in the size of synaptic vesicles, with a resulting loss of uniformity. Furthermore, electrophysiological analyses showed significant increases in the frequency, but not the amplitude, of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in response to Shh, providing functional evidence of the selective role of Shh in presynaptic terminals. Thus, we conclude that Shh signaling regulates the structure and functional properties of presynaptic terminals of hippocampal neurons.

mGRASP enables mapping mammalian synaptic connectivity with light microscopy.

The GFP reconstitution across synaptic partners (GRASP) technique, based on functional complementation between two nonfluorescent GFP fragments, can be used to detect the location of synapses quickly, accurately and with high spatial resolution. The method has been previously applied in the nematode and the fruit fly but requires substantial modification for use in the mammalian brain. We developed mammalian GRASP (mGRASP) by optimizing transmembrane split-GFP carriers for mammalian synapses. Using in silico protein design, we engineered chimeric synaptic mGRASP fragments that were efficiently delivered to synaptic locations and reconstituted GFP fluorescence in vivo. Furthermore, by integrating molecular and cellular approaches with a computational strategy for the three-dimensional reconstruction of neurons, we applied mGRASP to both long-range circuits and local microcircuits in the mouse hippocampus and thalamocortical regions, analyzing synaptic distribution in single neurons and in dendritic compartments.

An initial HOPS-mediated fusion event is critical for autophagosome transport initiation from the axon terminal.

In neurons, macroautophagy/autophagy is a frequent and critical process. In the axon, autophagy begins in the axon terminal, where most nascent autophagosomes form. After formation, autophagosomes must initiate transport to exit the axon terminal and move toward the cell body via retrograde transport. During retrograde transport these autophagosomes mature through repetitive fusion events. Complete lysosomal cargo degradation occurs largely in the cell body. The precipitating events to stimulate retrograde autophagosome transport have been debated but their importance is clear: disrupting neuronal autophagy or autophagosome transport is detrimental to neuronal health and function. We have identified the HOPS complex as essential for early autophagosome maturation and consequent initiation of retrograde transport from the axon terminal. In yeast and mammalian cells, HOPS controls fusion between autophagosomes and late endosomes with lysosomes. Using zebrafish strains with loss-of-function mutations in vps18 and vps41, core components of the HOPS complex, we found that disruption of HOPS eliminates autophagosome maturation and disrupts retrograde autophagosome transport initiation from the axon terminal. We confirmed this phenotype was due to loss of HOPS complex formation using an endogenous deletion of the HOPS binding domain in Vps18. Finally, using pharmacological inhibition of lysosomal proteases, we show that initiation of autophagosome retrograde transport requires autophagosome maturation. Together, our data demonstrate that HOPS-mediated fusion events are critical for retrograde autophagosome transport initiation through promoting autophagosome maturation. This reveals critical roles for the HOPS complex in neuronal autophagy which deepens our understanding of the cellular pathology of HOPS-complex linked neurodegenerative diseases.Abbreviations: CORVET: Class C core vacuole/endosome tethering; gRNA: guide RNA; HOPS: homotypic fusion and protein sorting; pLL: posterior lateral line; Vps18: VPS18 core subunit of CORVET and HOPS complexes; Vps41: VPS41 subunit of HOPS complex.

SLC26A4-AP-2 mu2 interaction regulates SLC26A4 plasma membrane abundance in the endolymphatic sac.

Decreased presence or activity of human SLC26A4 at the plasma membrane is a common cause of hearing loss. SLC26A4 (Pendrin) is necessary for normal reabsorption of endolymph, the fluid bathing the inner ear. We identified the µ2 subunit of adaptor protein 2 (AP-2) complex required for clathrin-mediated endocytosis as a protein-partner of SLC26A4 involved in regulating its plasma membrane abundance. We showed that, in the endolymphatic sac, where fluid reabsorption occurs, SLC26A4 is localized along the apical microvilli of mitochondria-rich cells, in contact with the endolymph, and associated with clathrin-coated pits where µ2 and AP-2 are present. Based on SLC26A4 structure, the elements involved in SLC26A4-µ2 interaction were identified and validated experimentally, allowing modeling of this interaction at the atomic level. Pharmacological inhibition of clathrin-mediated endocytosis led to an increased plasma membrane abundance of hemagglutinin-tagged SLC26A4 virally or endogenously expressed in mitochondria-rich cells. These results indicate that the SLC26A4-µ2 interaction regulates SLC26A4 abundance at the apical surface of mitochondria-rich cells.